The invention relates generally to acyl phosphate analogues, particularly to aspartyl phosphate analogues, as well as methods of their production and use.
Phosphorylation is a frequent covalent modification that proteins undergo in a post-translational process, and a fundamental dynamic event in a large array of cellular functions. Based on the phosphorylated amino acid residue, several groups can be classified (Hunter (1991) Methods in Enzymology 200, 3-37): 1) phosphoesters on serine, threonine and tyrosine; 2) phosphothioester on cysteine; 3) phosphoramidates on histidine, arginine and lysine; and 4) phosphate acid anhydrides on aspartate and glutamnate. Unlike the others, the last type of bond is highly unstablexe2x80x94the half-life of an acyl phosphate linkage is just hours under physiological conditions (Koshland (1951) J. Am. Chem. Soc. 74, 2286-2292; Di Sabato et al. (1961) J. Am. Chem. Soc. 83, 4400-4405). Protein phosphorylation on an aspartate residue occurs widely. For example, phosphorylation on aspartate residues occurs in two large protein familiesxe2x80x94response regulators and the haloacid dehalogenase (HAD) superfamily of hydrolases.
Response regulators, together with their cognate autokinases, dominate signal transduction in the bacteria (Nixon et al. (1986) Proc. Natl. Acad. Sci. USA 83, 7850-7854; Parkinson and Kofoid (1992) Annu. Rev. Genet. 26, 71-112; Stock et al (1995) in Two-component signal transduction, eds. Hoch and Silhavy, T. J. (ASM, Washington, D.C.), pp. 25-51; Mizuno et al. (1996) DNA Res. 3, 407-414.; Mizuno (1997) DNA Res. 4, 161-168) and are also found upstream of protein kinase cascades in eukarya (Ota et al. (1993) Science 262, 566-569; Maeda et al. (1994) Nature (London) 369, 242-245; Ruis and Schxc3xciller (1995) BioEssays 17, 959-965; Schaller (1997) Essays Biochem. 32, 101-111). Autokinase/response regulator pairs, which are referred to as xe2x80x9ctwo-componentxe2x80x9d systems, control bacterial cell division (Quon et al. (1996) Cell 84, 83-93), development (Fabret et al. (1999) J. Bacteriol. 181, 1975-1983), chemotaxis (Silversmith and Bourret (1999) Trends MicrobioL 7, 16-22), virulence (Dziejman and Mekalanos (1995) in Two-component signal transduction, eds. Hoch and Silhavy, (ASM, Washington, D.C.), pp. 305-317; Haldimann et al. (1997) J. Bacteriol 179, 5903-5913; Groisman (1998) BioEssays 20, 96-101; Novak et al. (1999) Nature (London) 399, 590-593), and the responses to many changes in nutrient availability (Ninfa et al. (1995) in Two-Component Signal Transduction, eds. Hoch and Silhavy (ASM, Washington, D.C.), pp. 67-88; Wanner (1995) in Two-Component Signal Transduction, eds. Hoch and Silhavy (ASM, Washington, D.C.), pp. 203-221). In general, phosphorylation of an aspartate residue in receiver domains of response regulators is used to modulate the function of their corresponding output domains (Ninfa and Magasanik (1986) Proc. Natl. Acad. Sci. USA 83, 5909-5913), many of which activate or repress transcription (Parkinson and Kofoid (1992) Annu. Rev. Genet. 26, 71-112; Stock et al. (1995) in Two-component signal transduction, eds. Hoch and Silhavy, T. J. (ASM, Washington, D.C.), pp. 25-51).
The structures of six unphosphorylated receiver domains have been determined (Stock et al. (1989) Nature (London) 337, 745-749; Volkman et al. (1995) Biochemistry 34, 1413-1424; Baikalov et al. (1996) Biochemistry 35, 11053-11061; Feher et al. (1997) Biochemistry 36, 10015-10025; Djordjevic et al. (1998) Proc. Natl. Acad. Sci. USA 95, 1381-1386; Sola et al. (1999) J. Mol Biol. 285, 675-687) and it has been shown that a substantial conformational change occurs upon phosphorylation in several cases (Drake et al. (1993) J. Biol. Chem. 268, 13081-13088; Lowry et al. (1994) J. Biol Chem. 269, 26358-26362; Nohaile et al. (1997) J. Mol. Biol. 273, 299-316). However, the lability of the acyl phosphate linkages in these domains (half lives of seconds to hours) (Parkinson and Kofoid (1992) Annu. Rev. Genet. 26, 71-112; Stock et al. (1995) in Two-component signal transduction, eds. Hoch and Silhavy, T. J. (ASM, Washington, D.C.), pp. 25-51) has hindered structural studies of their phosphorylated, active forms.
The response regulator NtC (nitrogen regulatory protein C), an enhancer-binding protein, functions as a molecular machine to activate transcription by the "sgr"54-holoenzyme form of RNA polymerase (Magasanik (1996) in Regulation of gene expression in Escherichia coli, eds. Linand Lynch (R. G. Landes Co., Austin), pp. 281-290; Rombel et al. (1998) Cold Spring Harbor Symp. Quant. Biol. 63, 157-166; Porter et al. (1995) in Two-component signal transduction, eds. Hoch and Silhavy, T. J. (ASM, Washington, D.C.), pp. 147-158). The NtrC protein of Salmonella typhimurium is composed of three functional domains (Drummond et al. (1986) EMBO J. 5, 441-447; North et al. (1993) J. Bacteriol. 175, 4267-4273; Morett and Segovia (1993) J. Bacteriol. 175, 6067-6074): an amino (N)-terminal receiver or regulatory domain that is phosphorylated on aspartate 54 (D54), a central output domain that hydrolyzes ATP and activates transcription, and a C-terminal DNA-binding domain. The central domain of NtrC apparently adopts a mononucleotide-binding fold characteristic of a large group of purine nucleotide-binding proteins (Osunan et al. (1997) Protein Sci. 6, 543-555; Li et al. (1999) J. Bacteriol. 181, 5443-5454). Phosphorylation of D54 allows NtrC to form large oligomers that are essential for ATP hydrolysis and hence transcriptional activation (Rombel et al. (1998) Cold Spring Harbor Symp. Quant. Biol. 63, 157-166; Porter et al. (1995) in Two-component signal transduction, eds. Hoch and Silhavy (ASM, Washington, D.C.), pp. 147-158; Porter et al. (1993) Genes Dev. 7,2258-2273; Wyman et al. (1997) Science 275, 1658-1661).
Despite the importance of the protein in transcriptional regulation, the receiver domain of NtrC (NtrCr) is the only such domain for which the structures of both the phosphorylated and unphosphorylated forms have been determined (Volkman et al. (1995) Biochemistry 34, 1413-1424). Although it was possible to maintain phosphorylated NtrCr (P-NtrCr) for long enough to allow determination of its structure by NMR spectroscopy, methods for doing so and for collecting and analyzing the necessary data were non-trivial.
The members of the HAD superfamily of hydrolases, which have diverse specificity (Koonin, E. V., et al., (1994) Journal of Molecular Biology 244, 125-132; Aravind, L., et al., (1998) Trends in Biochemical Sciences 23, 127-129), are examples of other important aspartate-phosphorylated proteins. Whereas 2-haloacid dehalogenase, the founding member of this large protein family, does not catalyze hydrolysis of phosphate substrates nor depend on Mg2+, most other members do. Among them, a phosphotransferase subgroup includes several phosphatases, such as phosphoserine phosphatase (PSP), and some forms of phosphomutases. Another subgroup is composed of the P-type ATPases. These proteins play a major role in ion transport across biological membranes (MacLennan et al. (2000) Nature 405, 633-634). Phospho-enzyme by intermediates have been detected in phosphotransferases (Seal, S. N., et al., (1987) Journal of Biological Chemistry 262, 13496-13500; Pirard, M., et al., (1997) Febs Letters 411, 251-254; Collet, J. F., et al., (1997) Febs Letters 408, 281-284; Collet, J. F., et al., (1998) Journal of Biological Chemistry 273, 14107-14112), and it is also well known that a phospho-intermediate forms during the action of P-type ATPases (MacLennan et al. (2000) Nature 405, 633-634) In both cases, phosphorylation requires Mg+ and occurs at a conserved aspartate residue.
Aluminofluoride and beryllofluoride are phosphate analogs for many purine nucleotide binding proteins, such as G proteins, FI-ATPase, myosin, and nitrogenase (see, e.g., Chabre (1990) Trends Biochem. Sci. 15, 6-10; Petsko (2000) Proceedings of the National Academy of Sciences of the United States of America 97, 538-540). In these cases beryllofluoride acts as a xcex3-phosphate analog, i.e., high affinity binding of the beryllofluoride analog requires the presence of a nucleoside diphosphate in the catalytic nucleotide site of the enzyme. Furthermore, these analogs tend to decrease or completely inhibit catalytic activity, i.e. ATPase or other activity of the modified enzyme is substantially decreased or undetectable.
There is a need for stable acyl phosphate analogues, particularly apartyl phosphate analogues. The present invention addresses this need.
The present invention features methods and compositions for production of persistent acyl phosphate analogues (e.g., aspartyl or glutamyl phosphate analogues) using beryllofluoride (BeFx), as well as polypeptides comprising such an acyl phosphate analogue and antibodies that specifically bind to these polypeptides. The invention further features methods of using BeFx analogues in screening assays to identify candidate agent compounds that modulate activity of polypeptides that normally exhibit activity due to the presence of an acyl phosphate linkage (e.g., a phosphorylated aspartate residue as in, e.g., polypeptides involved in signal transduction, polypeptides involved in ion transport across biological membranes, phosphotransferases, etc.). The BeFx polypeptide analogues can also be used to facilitate determination of the structure of the corresponding phosphorylated polypeptide and in rationale drug design.